Volume 141, Issue 5, Pages 1897-1906 (November 2011) A Proinflammatory Role for Interleukin-22 in the Immune Response to Hepatitis B Virus  Ye Zhang, Melissa A. Cobleigh, Jian–Qi Lian, Chang–Xing Huang, Carmen J. Booth, Xue–Fan Bai, Michael D. Robek  Gastroenterology  Volume 141, Issue 5, Pages 1897-1906 (November 2011) DOI: 10.1053/j.gastro.2011.06.051 Copyright © 2011 AGA Institute Terms and Conditions

Figure 1 IL-22 induces an acute phase–like response in the liver of HBV Tg mice but does not inhibit virus replication. (A) Groups of age-, sex-, and serum HBeAg-matched Tg mice (3 mice per group) were injected intravenously or intraperitoneally with a single 25-μg dose of IL-22 and analyzed 1 day after injection. HBV replication in the liver was measured by Southern blot (SB) analysis of relaxed-circle (RC) and single-stranded (SS) DNA replication forms and compared with levels in control animals that did not receive the cytokine. HBV 3.5 kilobase (kb) and 2.1 + 2.4 kb messenger RNA expression from total liver RNA was examined by Northern blot (NB) analysis and compared with the housekeeping gene GAPDH. (B) Intrahepatic expression of amyloid A (AA), haptoglobin (HB), and anti-chymotrypsin (α-CT) were measured by quantitative real-time reverse-transcription polymerase chain reaction. Results are displayed as fold differences relative to one control mouse and normalized to GAPDH expression. (C) Serum amyloid A (SAA) levels from the same mice were measured by enzyme-linked immunosorbent assay. (D) The level of serum alanine aminotransferase (sALT) was measured before and after injection. The data represent mean ± SD of 3 mice. Gastroenterology 2011 141, 1897-1906DOI: (10.1053/j.gastro.2011.06.051) Copyright © 2011 AGA Institute Terms and Conditions

Figure 2 IL-22 depletion does not affect IFN-γ–mediated inhibition of HBV replication but ameliorates subsequent liver damage following transfer of immunized splenocytes. (A) IL-22 expression in immunized splenocytes was measured by enzyme-linked immunosorbent assay after a 4-day incubation in vitro with or without HBsAg stimulation. Matched data points indicate splenocytes from an individual animal. (B) IL-22 expression from pooled total, CD4-depleted (CD4−; Miltenyi Biotec), or CD4-purified (CD4+) splenocytes. (C) HBV.CB6F1 mice (4 per group) were injected with 2 × 108 immunized splenocytes with or without anti–IL-22 antibody. Mice were killed at days 2 and 5 postinjection, and total hepatic DNA was analyzed for HBV replication by Southern blot (SB). Bands corresponding to integrated transgene (Tg), relaxed-circular (RC), and single-stranded (SS) HBV DNA replication intermediates are indicated. Northern blot (NB) was used for analysis of HBV 3.5 kilobase (kb) and 2.1 + 2.4 kb messenger RNA expression, normalized to GAPDH. Results were compared with control livers from another 4 matched Tg littermates injected with unimmunized splenocytes. (D) HBV.CB6F1 mice (4 per group) were injected with 2 × 108 CFSE-stained immunized splenocytes with or without anti–IL-22 antibody. The livers and spleens were harvested at day 2 after splenocyte transfer, and the percentages of CFSE+CD8+ cells in the liver and CFSE+ cells in the spleen were analyzed by flow cytometry. The data represent mean ± SD of 4 mice. (E) The mean serum ALT activity (±SD), measured 1 day preinjection and at the time the mice were killed 2 or 5 days after transfer, is indicated for each group. Gastroenterology 2011 141, 1897-1906DOI: (10.1053/j.gastro.2011.06.051) Copyright © 2011 AGA Institute Terms and Conditions

Figure 3 IL-22 neutralization reduces liver inflammation and pathology following transfer of immunized splenocytes. (A) Representative sections of liver from control HBV Tg mice given splenocytes from nonimmunized mice (upper panels), given immunized splenocytes alone (middle panels), and with anti–IL-22 antibody (lower panels) at day 2 (left) and day 5 (right) after administration. The overall morphology of control livers (upper panels) was normal. Mice with HBV-specific splenocyte-induced hepatic injury alone had more frequent foci of necrotic hepatocytes (arrowheads) and inflammation (asterisks) at day 5 (middle right panel) than day 2 (middle left panel). Mice with HBV-specific splenocyte-induced hepatic injury given anti–IL-22 antibody had fewer foci of necrosis and inflammation (lower panels) than mice not given anti–IL-22 antibody. Scale bars = 50 μm. (B) Histopathology scores for necrosis and inflammation were significantly reduced at day 5 in anti–IL-22 treated mice. Gastroenterology 2011 141, 1897-1906DOI: (10.1053/j.gastro.2011.06.051) Copyright © 2011 AGA Institute Terms and Conditions

Figure 4 Depletion of IL-22 blocks the recruitment of antigen-nonspecific cells into the liver. Livers from HBV Tg mice receiving splenocytes from nonimmunized mice (control) or immunized mice in the absence (-anti-IL-22) or presence (+anti-IL-22) of IL-22 antibody were weighed at the time the mice were killed, and IHLs were isolated from 2 liver lobes of a similar weight and analyzed by flow cytometry. The indicated numbers of total IHLs or splenocytes and cell subsets represent the absolute numbers in the liver or spleen, respectively. (A) Total IHL and splenocyte number, (B) CD4 T cells, (C) CD8 T cells, (D) natural killer cells, (E) neutrophils, and (F) B cells. Similar patterns were observed for natural killer T cells, lymphoid dendritic cells, myeloid dendritic cells, and macrophages (not shown). Gastroenterology 2011 141, 1897-1906DOI: (10.1053/j.gastro.2011.06.051) Copyright © 2011 AGA Institute Terms and Conditions

Figure 5 IL-22 depletion reduces chemokine expression in the liver. Expression of CXCL9 and CXCL10 in the liver of HBV Tg mice 2 days after receiving splenocytes from nonimmunized mice (control; n = 6) or immunized mice in the absence (-anti-IL-22; n = 8) or presence (+anti-IL-22; n = 6) of IL-22 antibody. RNA expression was measured by quantitative reverse-transcription polymerase chain reaction, and results are displayed as fold differences relative to the control group, normalized to GAPDH. Gastroenterology 2011 141, 1897-1906DOI: (10.1053/j.gastro.2011.06.051) Copyright © 2011 AGA Institute Terms and Conditions

Figure 6 Th17 cells and IL-22 expression in HBV-infected patients. (A) The percentage of circulating Th17 cells and (B) the concentration of IL-22 in serum were measured by flow cytometry or enzyme-linked immunosorbent assay in healthy controls (n = 16), patients with AHB (n = 16), CHB (n = 41), and asymptomatic HBV carriers (AsC, n = 20). Horizontal bars indicate the mean value of each subset. The individual frequency for each subject is shown. Significance was calculated using the Dunn's multiple comparison test. (C and D) Spearman correlation analysis of Th17 percentage with serum aspartate aminotransferase and ALT levels in 16 patients with AHB. Gastroenterology 2011 141, 1897-1906DOI: (10.1053/j.gastro.2011.06.051) Copyright © 2011 AGA Institute Terms and Conditions

Supplementary Figure 1 Gastroenterology 2011 141, 1897-1906DOI: (10.1053/j.gastro.2011.06.051) Copyright © 2011 AGA Institute Terms and Conditions